TRalpha1 mRNA Research Articles

Prenatal Ethanol Exposure Selectively Reduces the mRNA Encoding α‐1 Thyroid Hormone Receptor in Fetal Rat Brain

Some of the developmental defects characteristic of congenital or experimental hypothyroidism are also observed in children or experimental animals prenatally exposed to ethanol, suggesting that a subset of neurological defects attributable to ethanol exposure are produced by interfering with thyroid hormone action. In this article, we tested whether an ethanol treatment regimen known to produce neurological damage in rats can alter the expression of the mRNAs encoding the thyroid hormone receptor isoforms (TR alpha-1, TR alpha-2, and TR beta-1) in the fetal rat brain neocortex and hippocampus. Rats were fed an ethanol-containing diet beginning on gestational day (G) 6 and continuing until sacrifice on G15, G17, or G21; controls included animals pair-fed a liquid control diet or fed lab chow. Ethanol selectively reduced the expression of TR alpha-1 mRNA in the neocortex and hippocampus on G21, compared with pair-fed and control fetuses. In contrast, pair-feeding selectively reduced TR alpha-2 mRNA in both neocortex and hippocampus on G21, and increased TR beta-1 mRNA on G17. These data support the hypothesis that ethanol may interfere with thyroid hormone action during fetal brain development. In addition, these data indicate that ethanol and pair-feeding exert independent effects on thyroid hormone receptor expression in the developing brain.

Thyroid receptor plasticity in striated muscle types: effects of altered thyroid state.

This study examined nuclear thyroid receptor (TR) maximum binding capacity (Bmax), dissociation constant (Kd), and TR isoform (alpha1, alpha2, beta1) mRNA expression in rodent cardiac, "fast-twitch white," "fast-twitch red," and "slow-twitch red" muscle types as a function of thyroid state. These analyses were performed in the context of slow-twitch type I myosin heavy-chain (MHC) expression, a 3,5,3'-triiodothyronine (T3)-regulated gene that displays varying responsiveness to T3 in the above tissues. Nuclear T3 binding analyses show that the skeletal muscle types express more TRs per unit DNA than cardiac muscle, whereas the latter has a lower Kd than the former. Altered thyroid state had little effect on either cardiac Bmax or Kd, whereas hypothyroidism increased Bmax in the skeletal muscle types without affecting its Kd. Cardiac muscle demonstrated the greatest mRNA signal of TR-beta1 compared with the other muscle types, whereas the TR-alpha1 mRNA signals were more abundant in the skeletal muscle types, especially fast-twitch red. Hyperthyroidism increased the ratio of beta1 to alpha1 and decreased the ratio of alpha2- to alpha1+beta1-mRNA signal across the muscle types, whereas hypothyroidism caused the opposite effects. The nuclear T3 affinity correlated significantly with the TR-beta1 mRNA expression but not with TR-alpha1 mRNA expression. Collectively, these findings suggest that, despite a divergent pattern of TR mRNA expression in the different muscle types, these patterns follow similar qualitative changes under altered thyroid state. Furthermore, TR expression pattern cannot account for the quantitative and qualitative changes in type I MHC expression that occur in the different muscle types.

Functional retinoid and thyroid hormone receptors in human thyroid-carcinoma cell lines and tissues.

Thyroid carcinomas no longer accessible to radio-iodide or TSH-suppressive T4 therapy, due to loss of thyroid-specific functions, might be sufficiently re-differentiated by retinoic acid (RA) to be treated by conventional methods again. To help evaluate the feasibility of RA re-differentiation therapy in thyroid carcinomas, we examined the functionality of RA receptors (RARs/RXRs), central RA signal mediators, in human thyroid-carcinoma cell lines as model systems. [3H]-RA binding assays with nuclear extracts from follicular thyroid-carcinoma cell lines FTC-133 and -238 revealed high-affinity binding sites for RA. Electrophoretic mobility shift and super-shift assays using a DR2 ("direct repeat" 2) RA response element demonstrated DNA-binding of RARalpha, RARgamma, RXRalpha and RXRbeta in nuclear extracts of FTC-133 and anaplastic HTh74 cells. Use of a DR5 RA response element revealed no difference in DNA binding. In supershift assays with a DR4 T3 response element, we found DNA-binding by TRalpha1, TRalpha2, and TRbeta. Northern-blot analysis showed low expression of RXRbeta mRNA in FTC-133 and of TRalpha1 mRNA in FTC-133 and FTC-238 cells. Using RT-PCR, we detected mRNA for RARalpha, RARbeta, RARgamma, RXRalpha, and RXRbeta in the 4 cell lines and in human thyroid-carcinoma samples. RARbeta mRNA was reduced in FTC-238 cells and RXRbeta mRNA was decreased in anaplastic C643 cells and 9 of 12 tumor samples. Differential RA regulation of RA-receptor-mRNA expression was observed in the various cell lines. Thus, RA and T3 nuclear receptors are present in thyroid-carcinoma cell lines or tissues, albeit with cell-line and tumor-dependent variations; in the cell lines, they were shown to be functional with respect to DNA and/or ligand binding.

Thyroid hormone receptor isoform content in cultured type 1 and type 2 astrocytes.

Immunohistochemical studies previously reported from this laboratory showed that astrocytes in adult rat brain appear devoid of all thyroid hormone receptor (TR) isoforms. These findings, however, contrast with reports of measurable nuclear T3 binding in astrocytes in cell culture. To address this discrepancy, TR protein and messenger RNA (mRNA) content of type 1 and type 2 astrocytes in culture were assayed. Type 1 cells represent astrocytes present in brain in vivo. Type 2 astrocytes differentiate in culture from bipotential progenitor O-2A cells in the presence of serum. Under serum-free conditions, these progenitor cells differentiate into oligodendroglia. Total nuclear T3 binding capacity in both type 1 and type 2 astrocytes was approximately 3000 sites/cell. Northern blots showed the presence of mRNA for TRbeta1, TRalpha1, and TRalpha2 in type 2 cells but failed to reveal the presence of these mRNAs in type 1 astrocytes. Moreover, Northern blots also failed to reveal TRbeta2 mRNA in both type 1 and type 2 astrocytes. These findings, therefore, raised a question as to which receptor isoform was responsible for the nuclear binding capacity observed in type 1 astrocytes. As anticipated, immunocytochemical analysis demonstrated prominent nuclear signals for TRbeta1, TRalpha1, and TRalpha2 mRNA in type 2 astrocytes but failed to demonstrate TRbeta1, TRalpha1, or TRalpha2 in type 2 astrocytes. Application of RT-PCR, however, revealed the presence of low levels of TRbeta2 mRNA in type 1 astrocytes. When stained with a specific anti-TRbeta2 antiserum, both type 1 and type 2 astrocytes showed a strong fluorescent signal concentrated in the nucleus. These data indicate that under the special conditions of cell culture, expression of the TRbeta2 isoform in type 1 accounts for the measured nuclear T3 binding capacity.

Quantitation of endogenous thyroid hormone receptors alpha and beta during embryogenesis and metamorphosis in Xenopus laevis.

Greater than 90% of the endogenous thyroid hormone receptor proteins TR alpha and TR beta in tissues of Xenopus laevis comigrate with their respective in vitro synthesized counterparts, and these major components are not phosphorylated detectably. Maternally inherited TR alpha protein is stable through early embryogenesis during a time in which there is no detectable TR alpha mRNA synthesis. At stage 35 when TR alpha mRNA is first detectable, the inherited TR alpha protein is present at about 100 molecules/cell. TR alpha protein subsequently increases to levels of about 1500 and 6000 molecules/cell in tail and head regions, respectively, in stage 52 tadpoles. Even though TR alpha mRNA gradually increases during metamorphosis (from stage 52 to 62), TR alpha protein remains constant, suggesting strongly that post-transcriptional events control the ultimate levels of TR alpha protein. In contrast, there is no detectable TR beta protein (less than 100 molecules/cell) throughout embryogenesis until stage 52. Both TR beta mRNA and protein rise along with the increase in endogenous thyroid hormone, reaching a maximum at the climax of metamorphosis, when TR beta protein exceeds TR alpha protein in concentration. As with TR alpha protein, TR beta protein in tail is consistently about one-fourth that of TR beta protein in the head region. The number of TR alpha protein molecules in extracts of premetamorphic tadpoles and cultured cells grown in the absence of thyroid hormone fully accounts for all of the sites to which 125I-T3 bind. We interpret this to mean that TR alpha protein must be a necessary, if not sufficient, component in the pathway toward metamorphosis triggered by thyroid hormone and required for the phenomenon of competence in tissues and cells.

Distinct functions for thyroid hormone receptors alpha and beta in brain development indicated by differential expression of receptor genes.

Thyroid hormones are essential for correct brain development, and since vertebrates express two thyroid hormone receptor genes (TR alpha and beta), we investigated TR gene expression during chick brain ontogenesis. In situ hybridization analyses showed that TR alpha mRNA was widely expressed from early embryonic stages, whereas TR beta was sharply induced after embryonic day 19 (E19), coinciding with the known hormone-sensitive period. Differential expression of TR mRNAs was striking in the cerebellum: TR beta mRNA was induced in white matter and granule cells after the migratory phase, suggesting a main TR beta function in late, hormone-dependent glial and neuronal maturation. In contrast, TR alpha mRNA was expressed in the earlier proliferating and migrating granule cells, and in the more mature granular and Purkinje cell layers after hatching, indicating a role for TR alpha in both immature and mature neural cells. Surprisingly, both TR genes were expressed in early cerebellar outgrowth at E9, before known hormone requirements, with TR beta mRNA restricted to the ventricular epithelium of the metencephalon and TR alpha expressed in migrating cells and the early granular layer. The results implicate TRs with distinct functions in the early embryonic brain as well as in the late phase of hormone requirement.

A correlation of thyroid hormone receptor gene expression with amphibian metamorphosis.

The expression of the thyroid hormone (TH) receptor genes alpha (TR alpha) and beta (TR beta) in Xenopus laevis begins after the embryo hatches. The TR alpha mRNA increases throughout the premetamorphosis stage of tadpole development, is maximal by prometamorphosis, and falls after climax of metamorphosis to a lower level in frogs. The TR beta mRNA is barely detectable during premetamorphosis. In synchrony with the onset of endogenous TH synthesis by the thyroid gland (prometamorphosis), the level of TR beta mRNA rises in parallel with endogenous TH, reaching a peak at the climax of metamorphosis (stage 61) and drops to approximately 10% of its peak level after metamorphosis. As suggested by this correlation, exogenous TH up-regulates TR beta mRNA as much as 20-fold during premetamorphosis, whereas TH up-regulates TR alpha mRNA by approximately 2-fold during the same period. Up-regulation of TR beta mRNA is the earliest response to exogenous TH by competent tadpoles yet detected.